In collaboration with Simona Polo we have identified a novel ubiquitin-binding domain in myosin VI. We used NMR spectroscopy to solve the structure of the myosin VI ubiquitin-binding domain (MyUb). As part of this effort, we discovered myosin VI to prefer K63-linked chains, which are used for cell signaling rather than to signal for proteolysis. We solved the structure of MyUb complexed with K63-linked diubiquitin to find a unique recognition mode for this chain type. Moreover, we have mapped the region required for myosin VI interaction with endocytosis and autophagy adaptors, which overlaps with the MyUb. Myosin VI is expressed in humans as long or short isoforms that differ in the ubiquitin-binding region. We find that certain cancers preferentially express short myosin VI isoforms and that myosin VI knockdown in these cells restricts cell migration. We compared the structure of short and long myosin VI isoforms to find that short isoforms are missing a helix that interacts with MyUb. We are investigating the functional significance of this helical insert for interaction with various binding partners.